1. Field of the Invention
The field of this invention is high throughput screening assays for endocytosis.
2. Background
The ability to identify proteins involved in cellular pathways, map out the pathways and the proteins involved at the various stages and relate these to a phenotype has greatly expanded the need to identify the effect of various agents on the cellular phenotype and the target(s) for causing the effect. The interest in being able to identify compounds that are able to modulate the activity of a target and the increasing ability to prepare compounds has necessitated the development of protocols that allow for rapid and accurate evaluation of the activity of compounds, not only against the individual target. In order to have a successful assay, a significant number of requirements should be met. While ideally all of the requirements should be met, there will usually be compromises as to each of the requirements to provide a protocol that fulfills the overall needs of the user.
An efficient protocol will be accurate, repeatable, rapid, use conventional equipment, require as few steps as possible, particularly avoiding procedures that can introduce errors such as separations and washings, require readily available reagents, be adaptable for a number of determinations with different targets, require small volumes of sample and reagents, conveniently be performed in microtiter well plates, be robust and provide comparable results when performed in different venues. Fluorescent assays have many of these capabilities, but in many cases do not have the desired sensitivity, particularly where there is only one fluorescent molecule for each event. In some instances, it is desirable to have fusion proteins that can act as surrogates for the target proteins. The advent of green fluorescent protein and other fluorescent proteins has provided the opportunity to fuse the fluorescent protein with the target protein, but the quantum efficiency of these proteins is relatively low. Therefore, frequently to detect trafficking of the protein, it is expressed at high levels. These high levels can be deleterious to the cell, changing its phenotype and even leading to mortality.
Enzymes are attractive as labels, since they provide for amplification of signal by using a substrate whose product can be detected. Protocols can be devised that allow for a homogeneous assay without requiring separation and washing steps that introduce error into the results.
An important aspect of cellular activity is the protein population on the cell surface. Cellular membrane proteins fulfill many functions in transducing signals when ligand binds, acting as ion channels, binding to other proteins involving diapedesis, viral nucleic acid insertion, immune synapse, etc. For many receptors of clinical interest, upon binding to ligand, the cellular membrane receptor becomes endocytosed, so that the population at the surface may change in the presence of ligand or an agonist. When studying candidate compounds that may find use as drugs, acting as agonists or antagonists, one is interested in the amount of the cellular membrane protein that is endocytosed. By measuring the amount of protein that is endocytosed, one can measure the amount of binding of ligand or a mimetic ligand to the receptor, providing for a measure of the activity of the ligand or its mimetic analog.
Relevant Literature
Literature references of interest include Tsao and von Zastrow, 2000, J of Biol Chem 275, 11130–40; Neve, et al., 2001 Biochem Biophys Res Comm 280, 274–9; Liang and Huganir 2001 J of Neurochemistry 77, 1626–31; and Adie, et al., 2003 Assay and Drug Development Technologies 1, 251–9. Patent references of interest include US2002/0182643; US2004/0024005; US2004/0101912; and WO 02/033044.